Organic biofilm substrata as a microbial inoculum delivery vehicle for bioaugmentation of persistent organic pollutants in contaminated sediments and soils

ABSTRACT

A system and methods for removal of persistent organic pollutants (POPs) from an environment, where the system includes an inert and organic biofilm substrata as biofilm media for dual use: 1) inoculation of microorganisms to degrade POPs and 2) accumulation of POPs on the substrata, effective in maintaining bioavailable concentrations for sustaining microbial activity. Microorganisms capable of degrading or transforming POPs are actively associated with the substrata as a biofilm. Application of this delivery vehicle will enhance the microbial degradation of POPs, while simultaneously adsorbing hydrophobic POPs from the environment making them bioavailable for the microorganisms located in the formed biofilms and additionally lowering the aqueous concentration of POPs that have detrimental effects towards fish and mammals as they bioaccumulate through the food chain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/361,818, filed Jul. 6, 2010. The disclosure such application ishereby incorporated herein by reference in its entirety, for allpurposes.

GOVERNMENT RIGHTS IN THE INVENTION

This invention was made with government support under a grant awarded bythe United States Department of Defense's Strategic EnvironmentalResearch and Development Program Grant No. ER1502. The invention wasfurther made with government support under a grant awarded by theDepartment of Defense Office of Naval Research Grant No. N000140310035.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a persistent organic pollutant(POP)-degrading system containing a substratum and a biofilm containingPOP-degrading bacteria and where the system is capable of adsorbing anddegrading the POPs. The invention further relates to methods of treatingPOP-contaminated sediment or soil and methods of inoculatingmicroorganisms for POPs, using such a system. Still further, theinvention relates to a method of making such a system.

DESCRIPTION OF THE RELATED ART

Persistent organic pollutants (POPs) are pollutants present in theenvironment, globally dispersed throughout the ecosystem as a result ofcycling between air, water, and soil. Polychlorinated biphenyls (PCBs)are persistent organic pollutants that are hydrophobic and, as a resultof their hydrophobicity, PCBs bioaccumulate throughout the food chain byabsorption in the fatty tissue of animals and humans where they havebeen reported to act as endocrine disrupters and possible carcinogens.

Bioaugmentation is the introduction of natural microorganisms (or avariant thereof) a genetically engineered variant (inoculums) to treatcontaminated soil or water. At sites where soil and groundwater arecontaminated with POPs, bioaugmentation is used to ensure that the insitu microorganisms can completely degrade these contaminants tonon-toxic elements.

Bioaugmentation has been practiced for decades in agriculture andwastewater treatment and more recently for bioremediation of POPs incontaminated aquifers, waste streams or confined areas such as landfills(Aulenta, F., et al., (2006), Biodegradation 17(3):193-206;Robles-Gonzalez IV, et al., (2008), Microb. Cell Fact. 7:5). However,common for these approaches has been the demand for either a confinedsystem such as a slurry reactor or a liquid system such as groundwater.In these situations bioaugmentation has been based on liquid bacterialcultures as inoculum.

Degradation of less volatile POPs (e.g., PCBs, chlorobenzenes, etc.)occurring under anaerobic conditions in sediments and soil is a criticalprocess for their complete transformation to non-toxic forms. When grownin liquid culture, the density of many aromatic POP-transformingmicroorganisms such as the PCB dechlorinating bacteria is low, leadingto low efficiency of the progression of the reaction between themicroorganisms and the POPs. Specifically, in liquid culture, the POPsare not highly bioavailable to the microorganisms.

In sediment and soil, in situ microbial degradation of POPs underanaerobic conditions is a slow process due to the chemical andbiological stability of the contaminants, the low bioavailableconcentrations of individual contaminants and, in many cases, the lowabundance, diversity, and activity of naturally occurring POP degradingmicroorganisms. Therefore, it has been suggested that in situ biologicaltransformation of POPs in sediment and soil will not reduce theconcentration sufficiently within a reasonable time frame. Based on theconclusion that the affected sediment and soil sites are untreatable,the removal of POPs in situ has been achieved by removal of the affectedsediment and soil sites by dredging of the sites, and removal of thedredge spoil to contained locations such as landfills.

However, the action of removal of impacted sediments and soil can causeunwanted release of POPs into the environment. The physical disturbancedue to dredging will impact benthic organisms in the environmentdirectly and the concentration of POPs in the water phase will increasedue to re-suspension of sediment particles containing POPs. This willcause harm for benthic organisms and the surrounding environment sincethe contaminated sediment will be spread. Activated carbon has been usedfor sequestration of less volatile POPs such as PCBs and PAHs to preventthese contaminants from entering the water column in cases of dredgingor at heavily contaminated sites, where the solid-liquid equilibriumresults in the presence of POPs in the water phase. The results fromboth laboratory and field studies show that activated carbon is veryeffective in removing POPs from the water phase and thereby reducing thetoxicity towards benthic organisms (Sun X, Ghosh U. (2008), Environ.Toxicol. Chem., 27, 2287-2295.). In addition, since the activated carbonis mixed with the sediment (e.g., by injection or tilling), theparticles cannot be distinguished from sooth, black carbon and otherorganic particles that are naturally present in the sediment.

There therefore remains a need in the art for a system of POP reductionor removal effective in bioaugmentation of POP-contaminated sedimentsand soils, where the inoculum is not supplied as a liquid culture andwhere the system is useful both in confined systems and in situ.

SUMMARY OF THE INVENTION

The present invention relates to a system and method useful in reductionor removal of POPs from POP-contaminated sediment or soil, treatment ofcontaminated sediment or soil and inoculation of microorganisms forPOPs.

In one aspect the invention provides a system for at least partiallyreducing persistent organic pollutants (POPs) from an environment, thesystem comprising: an inert substratum effective to adsorb hydrophobicPOPs; and a biofilm on the substratum, wherein the biofilm comprises anactive inoculum.

In another aspect the invention provides a method of making a system forat least partially reducing POPs from an environment, the methodcomprising formation of a biofilm on a substratum effective to adsorbhydrophobic POPs, wherein the biofilm comprises an active inoculum.

In a still further aspect the invention provides a method of treating aPOP-containing environment, the method comprising administration of asystem to the POP-containing environment, the system comprising: aninert substratum effective to adsorb hydrophobic POPs; and a biofilm onthe substratum, wherein the biofilm comprises an active inoculum andwherein the system is effective to at least partially reduce POPs in thePOP-containing environment.

In yet another aspect the invention provides a method of reducing POPsin a locus containing the same, comprising introducing to said locus abiofilm comprising an active inoculum supported on a hydrophobicsurface.

In another aspect the invention provides a method of bioaugmenting anaerobic dechlorination system, the method comprising administration ofan anaerobic dechlorination system comprising: an inert substratumeffective to adsorb hydrophobic POPs and a biofilm on the substratum,wherein the biofilm comprises an active inoculum.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating the reductive dehalogenation of Aroclor1260 in Baltimore Sediments microcosms incubated with and without GAC,as detailed in Example 1 below.

FIGS. 2A and 2B are graphs demonstrating the PCB dechlorination activityof sediment collected from Baltimore Harbor, Md. in the presence/absenceof activated carbon showing effects of GAC on dechlorination productsfrom Aroclor 1260 as individual congeners (A) and congeners homologs(B).

FIG. 3 is a micrograph from a Confocal Laser Scanning Microscope showingan example of a biofilm forming on a GAC particle. (10 timesmagnification.)

FIG. 4 is a graph demonstrating the successful dechlorination ofPCB-contaminated sediment collected from Baltimore Harbor, Md. in thepresence of Dehalobium chlorocoercia, DF-1, illustrating the effects ondominant PCB congeners.

FIG. 5 is a graph demonstrating PCB analysis by homolog at day 0 (whitebars) and day 120 (black bars) after treatment as described in Example2, with (A) filter sterilized spent growth medium, (B) sterilized spentgrowth medium and GAC, (C) concentrated DF1 in growth medium inoculateddirectly into the sediment, and (D) concentrated DF1 adsorbed onto GAC.

FIG. 6 is a graph demonstrating the changes in mols Cl per mols PCB inmesocosms containing sediment contaminated with low levels of Aroclor1260 over time after treatment with an anaerobic PCB dechlorinator.

FIG. 7 is a graph with the enumeration of total dechlorinatingChloroflexi normalized to 16S rRNA gene copies/gram sediment at day 0,60 and 120.

FIG. 8 is a DHPLC community analysis of dechlorinating Chloroflexi 16SrRNA genes in mesocosms at day 0 (bottom trace) and day 120 (top trace):(A) spent growth media, (B) spent growth media+GAC, (C) DF1, and (D)DF1+GAC.

FIG. 9 is a graph illustrating DHPLC community analysis of dechlorinatorphylotypes in a mesocosm with DF-1.

FIG. 10 is a graph demonstrating the changes in absolute amount of PCBsin mesocosms containing sediment contaminated with low levels of Aroclor1260 over time after treatment with an anaerobic PCB dechlorinator andan aerobic degrader, as described in Example 4.

FIG. 11 is a graph demonstrating PCB analysis by homolog at day 0 (graybars) and day 120 (black bars) after treatment as described in Example4, with (A) spent growth medium and GAC, (B) GAC and lactate, (C) GAC,lactate and LB400, and (D) GAC, lactate, LB400 and DF-1.

FIG. 12 is a graph demonstrating the effect of GAC, DF-1 and LB400 onPCB congeners.

FIG. 13 is a graph illustrating DHPLC community analysis ofdechlorinator phylotypes in a mesocosm with DF-1 and LB400.

FIG. 14 is an illustrative model of a system of the invention, with asubstratum 1, active inoculum 2 adsorbed as a biofilm and adsorbed POPs3.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS THEREOF

The present invention relates to a system useful in reduction or removalof POPs from a POP-contaminated environment via bioaugmentation andmethods of making such a system. The invention further relates tomethods of treating a POP-contaminated environment employing such asystem. The invention further relates to methods of inoculatingmicroorganisms for POPs, employing such a system.

A POP-containing or POP-contaminated environment, as used herein is anyenvironment in which POPs are found to have accumulated. Environments inwhich a system of the invention are useful include, but are not limitedto soil or sediment present in agricultural or aquacultural systems,wastewater systems, water treatment systems or natural or man-madebodies of water such as harbors, bays, lakes, rivers, oceans and thelike. In various embodiments, a system of the invention is useful insitu, in such environments, or is useful in the treatment of soil orsediment retrieved from such environments.

“Persistent organic pollutants” or “POPs” as used herein, refer toorganic compounds resistant to normal, environmental degradation. POPsoften have low or medium volatility and low water solubility. Due tothese qualities, POPs are known to accumulate in various environments,both in natural, in situ environments, and in confined, closed orcontrolled environments, e.g., an aquaculture system. The less-volatilehydrophobic POPs often adsorb to sediment and/or soil particles of suchenvironments. Examples of POPs include: polyaromatic hydrocarbons(PAHs), polyhalogenated aromatic hydrocarbons, polycyclic aromatichydrocarbons, polychlorinated biphenyls, chlorinated aromatics,organochlorine pesticides, dioxins, benzofurans, polychlorinatedbiphenyls (PCBs), chlorobenzenes, chlorophenols, carbon tetrachloride,aldrin, chlordane, dichlorodiphenyl trichloroethane (DDT), dieldrin,endrin, heptachlor, hexachlorobenzene, mirex, toxaphene, polychlorinateddibenzo-p-dioxins (dioxins), polychlorinated dibenzofurans (furans) andthe like.

PCBs, as one type of POP, raise concerns due to their stability,toxicity and ability to bioaccumulate, as with other POPs. There are 209PCB congeners that have been identified and numbered. While particularexamples of PCBs as POPs are provided herein, the system and methods ofthe invention relate generally to reduction or removal of any POP with alow aqueous solubility and with a microorganism capable of selectivelyattacking that POP.

Microorganisms useful in the system and methods of the invention aremicroorganisms that can attack, degrade, reduce, transform or otherwiseaffect the POPs, including rendering them more subject to degradation bynative microorganisms.

By the present invention it was determined that the efficiency seen inliquid culture methods of administration of POP-degrading microorganismscould be improved by providing a hydrophobic surface to which the POPscan adsorb. Such adsorption to hydrophobic surfaces increases thebioavailable concentration of the POPs to the microorganisms. Therefore,the present invention provides a system for bioaugmentation, to enhancethe naturally occurring transformation of POPs in the environment usingefficient delivery vehicles for supply of a highly active inoculum.

An “active inoculum,” as used herein, refers to a microorganism added toa POP-contaminated environment which affects the POPs in a manner thatdegrades them and/or makes them more subject to degradation. In oneembodiment, the active inoculum is a POP-degrading bacteria or aPOP-transforming bacteria.

Microorganisms useful as an active inoculum in the system and methods ofthe invention may include, but are not limited to, dehalorespiringmicroorganisms, such as, Dehalococcoides spp., Dehalobium spp.,Desulfitobacterium spp., Desulfomonile spp., Geobacter spp. and PCBoxidizing bacteria, such as, Burkholderia spp., Rhodococcus spp.,Luteibacter spp., and Williamsia spp.

The substrata of the invention are solids, which may be constructed froma variety of organic materials into a variety of shapes and sizes. Forexample, activated carbon can be used, in a granular, powdered orotherwise useful form. The substratum is comprised of an inert substanceand does not limit the activity of the active inoculum. In a particularembodiment, the substratum provides a stimulatory effect to the activityof the active inoculum. In a particular embodiment, the substratacomprise granulated activated carbon (GAC). In a further embodiment, thesubstratum comprises any known or commercially available organicsubstratum.

The substrata contain a biofilm comprised of an active inoculum formedthereon. Microorganisms capable of degrading POPs will be activelyassociated with the substrata as a biofilm. In one embodiment of theinvention, the POP-degrading bacteria are surrounded by hydrophobiclayers that enable the individual bacterial cells to align in closeproximity to particles and thereby interact with hydrophobic POPs thatoften are adsorbed to surfaces in the aquatic environment due to theirhydrophobic nature. By supplying a hydrophobic surface as a growthmedium, the POP-degrading bacteria forms a biofilm on the substratum,where the biofilm has a much larger cell density than would be possibleto obtain in liquid cultures and therefore is very effective as inoculumfor bioaugmentation in the environment.

The system of the invention therefore provides an inert and organicbiofilm substratum as a biofilm media for dual use, including both: 1)inoculation of microorganisms to degrade persistent organic pollutants(POPs) and 2) accumulation of these POPs to maintain bioavailableconcentrations for sustaining microbial activity.

In one embodiment, the invention provides a system for reduction orremoval of POPs from POP-contaminated environment (e.g., aqueousenvironment containing sediment and/or soil), where the system comprisesan inert substratum and a biofilm comprising an active inoculum formedthereon, and the system is capable of adsorbing and degradinghydrophobic POPs from the environment in which the system is applied. Ina particular embodiment, the POPs are PCBs.

It was previously believed that an active inoculum would not function todegrade POPs in the presence of carbon due to reduced bioavailability tothe microorganism. Contrary to that commonly held assumption, thepresent invention demonstrates that use of carbon as a substrate for theactive inoculum and the POPs both permits and stimulates the degradationof POPs. The presence of the solid substrata in the system of theinvention allows for administration of the system and the incorporatedbiofilm to the environment to be treated in a solid form, as analternative to supplying POP-degrading organisms as liquid cultures forbioaugmentation. The system of the present invention provides faster andmore selective degradation of POPs, as compared to previous systems andmethods.

In one embodiment, cultured PCB dehalorespiring bacteria may be used asan active inoculum in a system of the invention, as exemplified inExample 2 herein. As seen in FIGS. 4-6, the dehalorespiring Dehalobiumchlorocoercia DF1 stimulated dechlorination of PCBs and resulted in asignificant decrease of PCBs in the environment. Results presented inFIG. 4 show that addition of DF-1 to PCB contaminated sediment using GACfor inoculating the dehalorespiring bacteria accelerated the reductivedechlorination of weathered PCB congeners even after 120 days ofincubation. The sustabinability of such system is shown in Example 3below.

In a further embodiment, a system of the invention provides reductivedechlorination of higher chlorinated congeners to lower chlorinatedcongeners. As demonstrated herein, bioaugmentation with thedehalorespiring bacterium DF-1 successfully stimulated the reductivedechlorination of sediment containing 1.3 ppm of weathered Aroclor 1260.Similarly highly chlorinated congeners that may be reductivelydechlorinated by a system of the invention include, but are not limitedto Aroclor 1016, 1221, 1232, 1242, 1248, 1254, 1258, 1260, 1262, 1268,and various commercial PCB mixtures, known under trade names such asAroclor, Inerteen, Pyranol, Abestol, Askarel, Bakola131, Chlorextol,Hydol®, Noflamol, Saf-T-Kuhl, and Therminol® in the United States,Kanechlor, Santotherm, and Pyrochlor in Japan, Askarel in the UnitedKingdom, Phenoclor, and Pyralene in France, Clophen in Germany, andFenclor in Italy.

A system of the invention, comprising substrata and a biofilm comprisingan active inoculum, is effective in bioaugmentation of a PCB or otherPOP-contaminated environment. A system of the invention must besustainable within the indigenous microbial community, must notinterfere with the bioavailability of the levels of PCB or other POPstargeted in the environment and must effectively disperse into theenvironment to be treated.

Bioavailability

Example 2 demonstrates that bioaugmentation with the dehalorespiringbacterium DF-1 successfully stimulated the reductive dechlorination ofsediment containing 1.3 ppm of weathered Aroclor 1260. Furthermore,addition of GAC, which increases the partition coefficient of the PCBbetween the sediment matrix and aqueous phase, had no inhibitory effecton dechlorination activity in bioaugmented mesocosms, as compared withno GAC treatment. As illustrated by FIG. 2A, the addition of GACpromoted a more extensive anaerobic microbial transformation ofcommercial complex PCB mixtures to less chlorinated congeners comparedwith sediment microcosms without GAC. This is contrary to earlierteachings that increasing the partition coefficient of PCBs with organiccarbon, which effectively reduces the bioavailability of PCBs tomacroorganisms, would also reduce the bioavaiability to micoroorganisms.It is further contemplated by the present invention that PCBdehalorespiring bacteria have mechanisms that enable them to compete forPCBs in environments containing high amounts of organic carbon.

In one embodiment, a system of the invention, comprising substrata and abiofilm comprising an active inoculum, is effective in stimulation ofreductive dechlorination of low levels of weathered PCBs in the 1-2 ppmrange even in the presence of a high background of organic carbon.

Sustainability

In one embodiment, a system of the invention includes an active incoulumthat effectively competes with bacteria present within the environmentto be treated, to effectively bioaugment the degradation system withinthe environment.

In an embodiment where the POP is a PCB, the active inoculum mustcompete with the indigenous community of non-dehalorespiring bacteriafor electron donors and nutrients, and with the indigenousdehalorespiring community for low levels of PCBs in order tosuccessfully bioaugment weathered PCB-impacted sediments. In Example 3,DF-1 was detected in bioaugmented sediment mesocosms after 120 daysindicating that bioaugmentation was competitive with a low concentrationof weathered Aroclor in a background of the indigenous sedimentcommunity. The total numbers of dehalorespiring bacteria decreased byapproximately half after 60 days to a steady state of 7-8×10⁵ cells pergram of sediment (FIG. 7), but community analysis showed that DF-1 wasretained as a predominant member of the dehalorespiring population (FIG.9). The total numbers of dehalorespiring phylotypes was 1-2 orders ofmagnitude lower than observed in prior reports of Aroclor 1260dechlorinating microcosms, but unlike the prior studies an electrondonor was not added and the lower steady state numbers appear to reflectlower concentrations of indigenous electron donor available in thesediment.

The sustainment of the dehalorespiring bacteria in the sedimentmesocosms without an exogenous electron donor is consistent with theability of dehalorespiring bacteria to outcompete hydrogenotrophicsulfate reducers, acetogens, and methanogens in the presence of limitedhydrogen concentrations. Indigenous phylotypes of dehalorespiringbacteria, with the exception of phylotype DEH10 had not been reportedpreviously in Baltimore Harbor sediment Aroclor 1260 enrichmentmicrocosms. However, in addition to supporting this indigenouspopulation of dehalorespiring bacteria, the results show that DF-1 wasable to compete successfully with the indigenous microbial populationeven with the lower background of electron donor.

Further, the detection of PCB congeners not resulting fromdechlorination of doubly flanked chlorines were observed. The detectionof indigenous dehalorespiring phylotypes throughout the incubationperiod in the current study (FIG. 8) support the conclusion that DF-1had a stimulatory effect on the indigenous dehalorespiring community.One possible explanation for this effect is “priming” by theaccumulation of dechlorination products from the initial activity ofhigh numbers of DF-1 inoculated into the sediments. Addition of PCBcongeners and analogs is known to stimulate the reductive dechlorinationof PCBs in lab studies and in field tests. Overall, the resultsdemonstrate that, in addition to initiating and directly dechlorinatingweathered PCBs, bioaugmentation with DF-1 had a synergistic effect onthe indigenous dehalorespiring community.

Bioaugmentation

There have been numerous reports on the potential of aerobicbioaugmentation with bacteria, fungi and plants, but these processeshave limited capacity to attack highly chlorinated congeners often foundin PCB impacted sites (Abraham, W. R., et al., Curr Opin Microbiol.,2002, 5, 246-253). Reductive dechlorination of higher chlorinated PCBcongeners has the potential to complement these processes but there havebeen very few studies to date describing the use of anaerobicbioaugmentation to stimulate in situ treatment of PCB impactedsediments.

Previous attempts to stimulate the reductive dechlorination of weatheredPCBs in sediment microcosms by bioaugmentation without adding ahalogenated congener as a primer were unsuccessful. There have beenrecent attempts to test the effects of bioaugmentation with purecultures of dehalorespiring bacteria. Krumins et al. (Krumins, V., WaterRes., 2009, 43, 4549-4558) reported enhanced reductive dechlorination ofweathered PCBs (ca. 2 ppm) in Anacostia River sediment microcosms afterbioaugmentation with D. ethenogenes strain 195 and May et al. (May, H.D., et al., Appl Environ Microbiol., 2008, 74, 2089-2094) reportedenhanced dechlorination of Aroclor impacted soil (4.6 ppm) afterbioaugmentation with DF-1.

In contrast to the prior studies where bioaugmentation with purecultures of dehalorespiring bacteria stimulated the reductivedechlorination of PCB by 0.2 Cl/biphenyl after 415 days and 0.35Cl/biphenyl after 145 days, respectively, bioaugmentation in the currentstudy stimulated the reductive dechlorination by 0.7 Cl/biphenyl afteronly 120 days. Furthermore, bioaugmentation results in the presentinvention stimulated 56% by mass reduction of penta- throughnona-chlorobiphenyls to mono- through tetrachlorobiphenyls, which aresusceptible to aerobic degradation, with no detectable activity inuntreated controls. The discrepancies in the rates and extent ofdechlorination could possibly occur due to a number of factors includingavailable nutrients, presence of inhibitory co-contaminants, thedehalorespiring strain used and the growth state and numbers of cellsused for bioaugmentation. Distribution of cells in the present inventionwas effective either by direct injection or on GAC particles. Theability to use a solid substrate, such as GAC, for inoculation of cellsprovides a method for dispersing cells in the field.

In one embodiment, a system of the invention, comprising substrata and abiofilm comprising an active inoculum, is effective in anaerobicreductive dechlorination of higher chlorinated PCB congeners anddemonstrate the ability to disperse within an environment to be treated.

In a further embodiment, the invention provides a method ofbioaugmentation of an aerobic biotransformation method by combining theaerobic biotransformation method with a system of the invention.Anaerobic bioaugmentation of the aerobic biotransformation method iseffective to achieve combined POP degradation. Such resultantdegradation is more complete than either of the aerobic method oranaerobic methods in the absence of one another. The aerobic andanaerobic methods may be carried out separately or concurrently.

Provided herein is a system effective in bioaugmentation for treatmentof PCB impacted sediments and other POP-contaminated environments. Inone embodiment, the system does not interfere with the bioavailabilityof the POPs, such that it is effective to treat low levels of a POP in acontaminated environment. In a particular embodiment, the system iseffective to treat low levels of weathered PCBs in sediment mesocosms.In another embodiment, the system includes substrata that do not inhibitthe activity of the active inoculum. In a particular embodiment, thesubstratum is GAC, effective to enhance the overall bioaugmentation, ascompared to administration without GAC. In another embodiment, thesystem includes an active inoculum that has sustained activity, suchthat the active inoculum has effective throughout the dechlorinationprocess of PCBs and the active inoculum has a positive synergisticeffect on the indigenous dehalorespiring population that contribute tothe process.

In selection of a system of the invention, it will be appreciated thatthe compatibility of the active inoculum, the environment to be treated,the indigenous bacterial population of the environment and the substratawill all be considered.

In a further embodiment, the invention provides a method of making asystem of the invention. Such method includes known methods forformation of a biofilm on an organic substratum, under conditions thatpermit formation of a biofilm on the substratum. A system of theinvention may be made in advance of use or may be made at the time ofuse. In a particular embodiment, the system is made at the site of anenvironment to be treated.

In one embodiment, the active inoculum is applied to the substratum byspray application. Such application methods are particularly useful whenthe substratum is in a powdered or otherwise non-granular form. Inanother embodiment, the active inoculum is applied to the substratum bysoaking and/or submersing the substratum into a cell culture containingthe active inoculum.

Because many POP transforming bacteria are hydrophobic, organicsubstrata such as activated carbon can be used to effectively harvestand concentrate microorganisms from culture by surface adsorptionwithout centrifugation or filtration. The organic substrata withadsorbed microbial catalysts as an active inoculum can then be useddirectly as inoculum in POP-contaminated sediments or soils.

Use of a system of the invention will enhance the microbial degradationof POPs, while simultaneously adsorbing hydrophobic POPs from theenvironment, making them bioavailable for the microorganisms located inthe formed biofilms and, additionally, lowering the aqueousconcentration of POPs that have detrimental effects towards fish andmammals as they bioaccumulate through the food chain.

Therefore, in one embodiment, the invention provides a method ofinoculating microorganisms in an environment to degrade POPs in thatenvironment by use of a system of the invention. When the system is usedas a microbial inoculum delivery vehicle, the biofilm bacteria willenhance the degradation of POPs, while the contaminants present in theenvironment that are hydrophobic will adsorb to the organic surface ofthe substrata, thus increasing the bioavailable concentration of thePOPs to the microorganisms and thereby enhancing the microbialdegradation of POPs.

In a further embodiment, the invention provides a method of treating aPOP-contaminated environment, e.g., aqueous environment containingsediment and/or soil, to reduce or remove POPs from the environment byuse of a system of the invention.

The methods of the invention comprising inoculation and POP removal orreduction may be performed separately or concurrently.

The methods of the invention include introduction of an inert organicsurface (substrata) where 1) the active inoculum of POP-degradingmicroorganisms are attached in biofilms and 2) the POP can adsorb onto.This two-phased approach provides an efficient and cost effective methodfor inoculation of microorganisms for bioaugmentation as well as keepingthe POPs out of the water phase. Inert organic compounds such asactivated carbon used for formation of the POP degrading biofilms aresimilar to naturally occurring organic compounds in the environment,making recovery of these compounds unnecessary by the end of thebioaugmentation period, since the biofilm coated compounds will blend inwith the naturally occurring organic particles.

It was previously believed that a solid structure based POP removalsystem was not feasible in an in situ environment, largely because anumber of factors, such as predation, competition and sorption, conspireagainst bioaugmentation in non-confined systems. The system and methodsof the invention, however, overcome these issues, as organic substrata(such as activated carbon) have inherently high affinities forsimultaneous attraction of POP degrading biofilm forming bacteria andadsorption of POPs, which ensures close proximity to bioavailableelectron acceptors. As such, the structure of the system of theinvention (i.e., the presence of both solid substrate and biofilmelements) enables implementation of a two-phased approach, where organiccompounds are applied as substratum for biofilm formation and subsequentdelivery vehicles for bioaugmentation of POPs in contaminated sedimentsand soils.

The advantages and features of the invention are further illustratedwith reference to the following examples, which are not to be construedas in any way limiting the scope of the invention but rather asillustrative of embodiments of the invention in specific applicationsthereof.

Example 1 Organic Substrata as Microbial Inoculum Delivery Vehicles forBioaugmentation of POP in Contaminated Sediments and Soils

Absorbents such as granular activated carbon (GAC) have been used tosequester aromatic POPs in sediments to minimize their interaction withthe biological food chain. However, it has been assumed that whensequestered from the biological food chain the POPs would no longer bebioavailable to microbial transformation. FIG. 1 shows that the rates ofmicrobial reductive dechlorination of PCB in Baltimore harbor sedimentswere not significantly impacted by addition of GAC to contaminatedsediments. Furthermore, the results also indicate that addition of GACresults in a more complete reductive dehalogenation of Aroclor 1260based on higher occurrence of mono- and di-chlorinated homologs asprecuts with GAC compared with a higher occurrence of tri-, tetra andpenta-chlorinated homologs in cultures without GAC (FIG. 2). In FIG. 2it is seen that dechlorination activity occurred in all microcosmsindependent of activated carbon, but that in the presence of activatedcarbon a higher proportion of PCBs were transformed into lesschlorinated congeners that are accessible for aerobic degradation andsubsequently complete mineralization.

The results are consistent with GAC promotion of the microbial process,possibly by accumulating PCB to kinetically saturating concentrations inthe immediate proximity of the microbial biofilms. An example of amicrobial biofilm forming on a GAC particle is shown in FIG. 3, wherebiofilm bacteria were hybridized with the general probe for BacteriaEUB338 and attached to sediment particles mixed with activated carbon(10 times magnification).

Example 2 Simulation of In Situ Bioaugmentation of POP in ContaminatedSediments and Soils

The effectiveness of using GAC for inoculating PCB dechlorinatingmicroorganisms into PCB impacted sediments was successfully tested withDehalobium chlorocoercia, DF-1. Sediments and water retrieved from a PCBcontaminated site in the Baltimore Harbor were incubated with DF-1 usingGAC as a carrier, where the incubation was at room temperature for 120days.

Dehalobium chlorocoercia DF1 was grown anaerobically in estuarinemineral medium (ECl) as described previously (Berkaw, M., et al., ApplEnviron Microbiol. 1996, 62, 2534-2539; Wu, Q., et al. Environ. Sci. &Technol., 2002, 36, 3290-3294.) Sodium formate was added as the electrondonor at a final concentration of 10 mM and PCB 61 (2,3,4,5-PCB) wasadded in acetone (0.1% v/v) at a final concentration of 173 μM.Desulfovibrio sp. extract was added as a growth factor at a finalconcentration of 1% (v/v). Titanium(III) nitrilotriacetate (0.5 mM,TiNTA) was used as a chemical reductant to remove oxygen from themedium. D. chlorocoercia DF-1 was routinely grown in 50 ml of medium in160-ml serum bottles sealed with 20-mm Teflon-coated butyl stoppers(West Co., Lionville, Pa.). All cultures were incubated statically at30° C. in the dark. Growth was monitored by gas chromatographic analysisof PCB 61 dechlorination to PCB 23 (2,3,5-PCB) and by quantitative PCRof 16S rRNA gene copies (described below).

Mesocosm Experiments

Mesocosms were prepared in glass 2 liter TLC tanks (Fisher Scientific).PCB impacted sediments were sampled on 14 May 2009 from the NorthwestBranch of Baltimore Harbor (BH) with a petite Ponar grab sampler at39°16.8_N, 76°36.2_W and stored in the dark under nitrogen for 19 daysat 4° C. prior to use. Sediment was homogenized anaerobically bystirring in an anaerobic glove bag and 1 liter of the homogenizedsediment was added to each mesocosm tank with 2 cm of indigenous waterabove the sediment surface. A glass plate covered each mesocosm tominimize evaporation with a 1 cm gap on one end for air exchange. Waterlost due to evaporation was periodically replenished with deionizedwater to maintain the original salts composition and osmolarity of theharbor water.

The bioaugmentation inoculum was prepared using ten 50 ml cultures ofDF1 grown until approximately 50% of PCB 61 was dechlorinated to PCB 23.The cultures were transferred into 250 ml Oak Ridge bottles in ananaerobic glove box and sealed under a nitrogen-carbon dioxide (4:1)atmosphere. The bottles were centrifuged at 22,000×g for 30 min,decanted, and the pellets were pooled by resuspension in 50 ml ofsterile ECl medium. The concentration of pooled DF1 was approximately5×10⁷ 16S rRNA gene copies per ml. Spent medium supernatant was preparedby passing culture supernatant through a 0.22 micron filter (Millipore,www.millipore.com) to remove residual DF1 cells. Mesocosms were amendedwith one of 4 treatments in an anaerobic glove box:

(1) 20 ml of concentrated DF1 (about 5×10⁷ cells per ml);

(2) 20 ml of spent growth media;

(3) 20 ml of concentrated DF1 adsorbed to 25 g granulated activatedcarbon for 1 hour; or

(4) 20 ml of spent growth media adsorbed to 25 g activated carbon for 1hour.

No exogenous electron donor was added. Mesocosms were homogenized bystirring with a Teflon spoon, then removed from the anaerobic glove boxand incubated at 23° C. in the dark. Mesocosms were periodically sampledby taking 6 cm deep cores using a 5 ml syringe barrel with the end cutoff. Triplicate cores (5 ml) were sampled for each timepoint using arandom sampling grid and homogenized prior to analysis for PCBs and DNAas described below.

DNA Extraction

DNA was extracted by adding 0.25 g of sediment from each sample core toa PowerBead microfuge tube of a Power Soil DNA Isolation Kit (MOBIOLaboratories, Inc. www.mobio.com). The PowerBead tubes were mixed byhand prior to 30 s of bead beating at speed “4.5” using a FastPrep120(Q-Biogene, 8 CA). Total DNA was then isolated from the PowerBead tubesaccording to the manufacturer's directions. DNA was eluted in 100 μl ofTE buffer and quantified with a NanoDrop 1000 Spectrophotometer(ThermoScientific). All DNA samples were diluted to 2 ng/ul in TE bufferprior to analysis by qPCR or DHPLC.

Enumeration of PCB Dehalorespiring Bacteria by Quantitative PCR

The quantification of Chloroflexi 16S gene copies in each subcore wasperformed by quantitative PCR using iQ SYBR green supermix (Bio-RadLaboratories, Hercules, Calif.) and gene-specific primers for the 16SrRNA gene of the dechlorinating Chloroflexi (348F/884R) (Fagervold, S.K., et al. Appl. Environ. Microbiol. 2005, 71, 8085-8090.). Each samplemixture had a 25-μl reaction volume containing 1× iQ SYBR greensupermix, forward and reverse primers at a concentration of 500 nM, and1 μl of the prepared DNA. PCR amplification and detection were conductedin an iCycler (Bio-Rad Laboratories, Hercules, Calif.). Quantitative PCRconditions were as follows: initial denaturation for 15 min at 95° C.followed by 35 cycles of 30 s at 95° C., then 30 s at 61° C., then 30 sat 72° C. One copy of the gene per genome was assumed based on thegenomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoidessp, strain CBDB1(Seshadri, R., et al., Science. 2005, 307, 105-108;Kube, M. et al., Nat. Biotech., 2005, 23, 1269-73). QPCR data wasanalyzed with the MJ Opticon Monitor Analysis Software v3.1 and comparedto a standard curve of bona fide DF1 348F/884R 16S rRNA gene product.Amplification efficiencies were 80% or greater.

Community Analysis of PCB Dechlorinating Bacteria by Denaturing HPLC

Denaturing HPLC (DHPLC) analyses were performed using a WAVE 3500 HTsystem (Transgenomic, Omaha, Nebr.) as described previously (Kjellerup,B. V., et al., Environ Microbiol., 2008, 10, 1296-1309) except that theinstrument was equipped with a florescence detector (excitation 490 nm,emission 520 nm). The primer set 348F/884R was used for specific PCRamplification of 16S rRNA genes from dechlorinating bacteria within theChloroflexi (Fagervold, S. K., et al. Appl. Environ. Microbiol. 2005,71, 8085-8090.). DNA was amplified by PCR in 50 μl reaction volumes asdescribed previously and PCR products of the correct length wereconfirmed by electrophoresis using a 1.5% agarose gel. The 16S rRNA genefragments were analyzed in a 10 μl injection volume by DHPLC with aDNASep® cartridge packed with alkylated nonporouspolystyrene-divinylbenzene copolymer microspheres for high-performancenucleic acid separation (Transgenomic, Omaha, Nebr.). The oventemperature was 63.0° C. and the flow rate was 0.5 ml min⁻¹ with agradient of 55%-35% Buffer A and 45%-65% Buffer B from 0-13 minutes.Analysis was performed using the Wavemaker version 4.1.44 software.Individual peaks were eluted for sequencing and collected with afraction collector based on their retention times. Prior to sequencingindividual fractions were dried, dissolved in 15 μl nuclease free waterand re-amplified following the protocol described above. PCR productswere confirmed by DHPLC to ensure that only one species was present,then purified subsequently by electrophoresis in a 1.5% low melt agarosegel purification of excised fragments using Wizard® PCR Preps DNAPurification Resin/A7170 (Promega Corp., Madison, Wis.).

DNA Sequencing and Analysis

DHPLC fractions were sequenced in the 5′ and 3′ direction with 250 pM ofprimer 348F or 884R and 5% DMSO to reduce effects from potentialsecondary structure using the BigDye® Terminator v3.1 (AppliedBiosystems, Foster City, Calif.) kit following manufacturer'sinstructions. Sequencing of purified DNA was performed on an ABI 3130 XLautomated capillary DNA sequencer (Applied Biosystems, CA). At least 468nts of sequence from each phylotype were obtained in this manner.Sequence similarities were analyzed using the Basic Local AlignmentSearch Tool (BLAST). A phylogenetic tree was drawn using the TreeBuilder software found at the Ribosomal Database Project(http://rdp.cme.msu.edu/index.jsp).

PCB Extraction

Sediment samples were extracted using an Accelerated Solvent Extractor(Dionex) following EPA method 3545. Approximately 5 grams wet weightsediment was dried with pelletized diatomaceous earth at roomtemperature in a desiccator containing CaCl₂.2H₂O. The dried sediment(approximately 1 g) was transferred to an 11 ml stainless steelextraction cell containing 0.6 g Cu and 2.4 g fluorosil between twocellulose filters on the bottom of the cell and the remaining cellvolume was filled with anhydrous Na₂SO₄. To correct for extractionefficiency, 10 μl of a 400 μg l⁻¹ solution of PCB 166 in hexane waspipetted on top of the Na₂SO₄. The sample containing the surrogate wasextracted with approximately 20 ml of hexane at 100° C. and purged with1 MPa nitrogen. The sample was evaporated to a final volume of 1 ml at30° C. under nitrogen using a N-EVAP 111 nitrogen evaporator(Organomation Associates, Inc. Berlin Mass., US). Before PCB analysis,10 μl of PCB 30 and PCB 204 (400 μg l⁻¹ each in acetone) was added tothe sample as internal standards.

PCB Analysis

PCB congeners were analyzed using a Hewlett-Packard 6890 series II gaschromatograph (GC) with a DB-1 capillary column (60 m by 0.25 mm by 0.25μm; JW Scientific, Folsom, Calif.) and a ⁶³Ni electron capture detectorby a modified method of EPA 8082. PCB congeners in a mixture containing250 μg l⁻¹ Aroclor 1232, 180 μg l⁻¹ Aroclor 1248 and 180 μg l⁻¹ Aroclor1262 were quantified with a 10-point calibration curve using PCB 30 andPCB 204 as internal standards. Individual congeners and respectiveconcentrations were obtained from Mullins et al (Mullins, P. H., et al.Microbiol., 1995, 141, 2149-2156.). Fifty-five additional congeners notpresent in the Aroclor mixture that were potential dechlorinationproducts were added to the calibration table containing the Aroclorcongeners. The additional congeners were quantified with 10-pointcalibration curves at concentrations of 2, 5, 10, 20, and 40 μg l⁻¹ (induplicate) for the low range calibration and 40, 100, 200, 400, and 800μg l⁻¹ (in duplicate) for the high range calibration. Using thisprotocol 173 congeners were resolved in 130 individual peaks (notincluding the standards PCB 30 and PCB 204 and the surrogate PCB166).Co-eluting peaks were indicated as multiple congeners. The finalconcentration of individual congeners in samples was corrected for therecovery efficiency of the surrogate (typical recovery efficiencies were75% or greater).

Effects of Treatments on Reductive Dechlorination of Weathered PCBs

BH sediment mesocosms were bioaugmented with dehalorespiring D.chlorocoercia DF-1 to a final concentration of approximately 5×10⁵ cellsml⁻¹ and the PCB congeners were monitored over 120 days. In mesocosmsbioaugmented directly and with GAC as a solid substrate, there was a netdecrease in the total amounts of penta- through octa-PCBs and a netincrease in the total amount of tetra- and tri-PCBs over the course of120 days (FIG. 5). There was no obvious change in homolog distributionin non-bioaugmented mesocoms treated with spent medium or spent mediumand GAC. Bioaugmentation both directly and with GAC as a carriersubstrate resulted in 0.6 to 0.7 mol Cl per biphenyl dechlorinated,respectively, after 120 days (FIG. 6). Bioaugmentation with GAC had aslightly greater rate of dechlorination (0.0067 Cl/biphenyl/day)compared with direct injection (0.0041 Cl/biphenyl/day). A t-Test(two-sample assuming unequal variances, α=0.05) showed a significantdifference in dechlorination rates between the bioaugmentationtreatments with a P-value of 0.041 (df=4).

Single congener analysis of mesocosms bioaugmented with DF1 resulted ina significant decrease in higher chlorinated congeners and correspondingincrease in less chlorinated congeners (FIG. 4). The total mol sum ofpredicted substrates and products was 1.26±0.307×10⁻⁹ mol per gramsediment at day 0 and 1.02±0.639×10⁻⁹ mol per gram sediment at day 120.The amount of substrate congeners decreased from 1.00±0.228 to0.291±0.282×10⁻⁹ mol per gram sediment and the amount of productcongeners increased from 0.256±0.0786 to 0.729±0.356×10⁻⁹ mol per gramsediment. The difference in mol PCB might have resulted from the absenceof possible dechlorination products in the GC method or aerobicdegradation by indigenous microorganisms. Strikingly, PCB 194 decreasedfrom about 23 to 8 ppb, and PCB 133, the predicted product of the doubleflanked reductive dechlorination of PCB 194 was found to increase fromabout 1 to 12 ppb over 120 days. Terminal products of double flankedreductive dechlorination of PCB 180 (PCB153) and PCB 174 (PCB135) weredetected after 120 days, however, these products did not accumulate tosubstantial amounts. Furthermore, predicted products of doubly flankedreductive dechlorination of the remaining octa- and hepta-chlorinatedcongeners were not detected and the less chlorinated congeners that didaccumulate were not products resulting from dechlorination of doubleflanked chlorines. Since DF-1 can only reductively dechlorinate doublyflanked chlorines the dechlorination products result from enhancedactivity by the indigenous population of dehalorespiring bacteria.Addition of cell free medium used to grow DF-1 did not stimulate PCBdechlorination, indicating that the enhanced activity by indigenousmicroorganisms was the direct result of bioaugmentation with DF-1 cellsand did not result from “priming” by residual PCBs or biostimulation bythe medium.

Example 3 Sustainability of D. Chlorocoercia DF1 after Bioaugmentation

To determine whether DF-1 was sustainable in the presence of relativelylow PCB concentrations and the indigenous general microbial community ofdehalorespiring and non-dehalorespiring bacteria within the sediment,dehalorespiring microorganisms were enumerated during growth based onthe number of 16S rRNA gene copies per gram dry sediment. In bothbioaugmented mesocosms the numbers of dehalorespiring bacteria wasinitially about 2-fold higher then in untreated mesocosms (1.3×10⁶compared to about 6.0×10⁵ copies per gram, respectively) indicating thatDF-1 accounted for approximately half the total population ofdehalorespiring bacteria. A Student's t-Test (two-sample assumingunequal variances, α=0.05) showed a significant difference betweeninitial 16S rRNA gene copy numbers between treatments with a two-tailP-value of 0.01 (df=3). The total 16S rRNA copy numbers in treatedmesocosms decreased by 60 days before reaching an apparent steady statefor the 120 day incubation period (FIG. 7). However, for bioaugmentationtreatments by both direct injection and on GAC substrate, the totalnumber of dehalorespiring microorganisms remained nearly 2-fold higherin the bioaugmented mesocosms compared with the untreated mesocosms(about 8.0×10⁵ compared to about 4.5×10⁵ copies per gram at day 120,respectively). The results indicate that total number of dehalorespiringmicroorganisms in the mesocosms bioaugmented with DF1 were maintained athigher numbers during active dechlorination of the weathered Aroclorcompared with non-bioaugmented controls.

Seven predominant dehalorespiring phylotypes were detected by DHPLC inthe BH sediment mesocosms. The community was generally similar betweenmesocosms, with the exception of DF1, which was only detected inbioaugmented mesocosms, and did not change greatly over 120 days (FIG.8). The putative DF1 fraction was collected, sequenced, and found to be100% identical to DF1. One phylotype present at time zero (BH 4) was100% identical to phylotype DEH10, previously detected BH sedimentmicrocosms (Fagervold, S. K., et al. Appl. Env. Microbiol. 2007, 73,3009-18; Fagervold, S. K., et al. Appl. Env. Microbiol. 2005, 71,8085-90.), but no other previously reported BH phylotypes were detected.Aside from the phylotype indentified as DEH10, the other dehalorespiringChloroflexi did not group in the Dehalococcoides clade.

Example 4 Simulation of In Situ Bioaugmentation of POP in ContaminatedSediments and Soils

Sediments retrieved from a PCB contaminated site in the Baltimore Harborwere simultaneously bioaugmented with aerobic PCB degrader Burkholderiaxenovorans LB400 and anaerobic PCB dechlorinator DF1 inoculated asbiofilm on GAC. Lactate was added as electron donor.

Individual mesocosms were prepared with 1 liter of sediment and 5-8 ppmof weathered Aroclor 1260 and were inoculated with one of the following:

spent growth medium and 1% GAC;

lactate and 1% GAC;

LB400 and 1% GAC and lactate; and

LB400 and DF-1 and 1% GAC and lactate.

Results in FIG. 10 provide data for days 0 to 90 after inoculation,which demonstrate significant dechlorination and degradation of PCBafter bioaugmentation with both the anaerobe and aerobe together usingGAC. It is seen that there was a net decrease of mono- through nona-PCBsand that 75% of the PCBs were degraded in 90 days. Addition of GAC didnot have an adverse effect on the dechlorination and/or degradation.FIG. 11 provides the data for PCB analysis by homolog at day 0 (graybars) and day 60 (black bars) after treatment with (A) spent growthmedium and GAC, (B) GAC and lactate, (C) GAC, lactate and LB400, and (D)GAC, lactate, LB400 and DF-1. FIG. 12 illustrates that the addition ofDF-1 and LB400 together resulted in the degradation of higher and lowerchlorinated PCBs without net accumulation of less chlorinated PCBdechlorination products. FIG. 13 shows the presence of DF-1 at both days0 and 90. It is contemplated that incubation for 120 days will result incontinued dechlorination and degradation of the PCBs.

While the invention has been has been described herein in reference tospecific aspects, features and illustrative embodiments of theinvention, it will be appreciated that the utility of the invention isnot thus limited, but rather extends to and encompasses numerous othervariations, modifications and alternative embodiments, as will suggestthemselves to those of ordinary skill in the field of the presentinvention, based on the disclosure herein. Correspondingly, theinvention as hereinafter claimed is intended to be broadly construed andinterpreted, as including all such variations, modifications andalternative embodiments, within its spirit and scope.

1. A system for at least partially reducing persistent organicpollutants (POPs) from an environment, the system comprising: an inertsubstratum effective to adsorb hydrophobic POPs; and a biofilm on thesubstratum, wherein the biofilm comprises an active inoculum.
 2. Thesystem of claim 1, wherein the POPs are polychlorinated biphenyls(PCBs).
 3. The system of claim 1, wherein the substratum is an organicsubstance.
 4. The system of claim 1, wherein the substratum is granularactivated charcoal.
 5. The system of claim 1, wherein the activeinoculum is a POP-degrading bacteria or a POP-transforming bacteria. 6.The system of claim 1, wherein the active inoculum is Dehalobiumchlorocoercia DF-1.
 7. A method of making a system for at leastpartially reducing persistent organic pollutants (POPs) from anenvironment, the method comprising formation of a biofilm on asubstratum effective to adsorb hydrophobic POPs, wherein the biofilmcomprises an active inoculum.
 8. The method of claim 7, whereinformation of the biofilm on the substratum comprises harvestingmicroorganisms from culture.
 9. The method of claim 7, wherein thesubstratum is an organic substance.
 10. The method of claim 7, whereinthe substratum is granular activated charcoal.
 11. A method of treatinga persistent organic pollutant (POP)-containing environment, the methodcomprising administration of a system to the POP-containing environment,the system comprising: an inert substratum effective to adsorbhydrophobic POPs; and a biofilm on the substratum, wherein the biofilmcomprises an active inoculum, wherein the system is effective to atleast partially reduce POPs in the POP-containing environment.
 12. Themethod of claim 11, wherein the POPs are polychlorinated biphenyls(PCBs).
 13. The method of claim 11, wherein the environment is soil orsediment.
 14. The method of claim 13, wherein the soil or sediment is insitu.
 15. The method of claim 13, wherein the soil or sediment is in aclosed or confined system.
 16. The method of claim 15, wherein theclosed or confined system is an aquaculture system.
 17. The method ofclaim 11, wherein the substratum is an organic substance.
 18. The methodof claim 11, wherein the substratum is granular activated charcoal. 19.The method of claim 11, wherein the active inoculum is a POP-degradingbacteria or a POP-transforming bacteria.
 20. The method of claim 11,wherein the active inoculum is Dehalobium chlorocoercia DF-1.
 21. Amethod of reducing persistent organic pollutants (POP)s in a locuscontaining same, comprising introducing to said locus a biofilmcomprising an active inoculum supported on a hydrophobic surface. 22.The method of claim 21, wherein the POPs are PCBs.
 23. The method ofclaim 21, wherein the substratum is an organic substance.
 24. The methodof claim 21, wherein the substratum is granular activated charcoal. 25.The method of claim 21, wherein the active inoculum is a POP-degradingbacteria or a POP-transforming bacteria.
 26. A method of bioaugmentingan aerobic dechlorination system, the method comprising administrationof an anaerobic dechlorination system comprising: an inert substratumeffective to adsorb hydrophobic POPs; and a biofilm on the substratum,wherein the biofilm comprises an active inoculum.
 27. The method ofclaim 26, wherein the administration of the anaerobic dechlorinationsystem is performed concurrently with the dechlorination of the aerobicdechlorination system.